Apoptosis or programmed cell death (PCD) is a genetically regulated mechanism of active cell death observed during development and disease and is required for homeostasis in multicellular organisms. There is a distinct and precisely localized control over the fate of specific cells that undergo apoptosis in a mixed cell population. Apoptosis is a selective process for self-elimination of damaged or undesired cells in various biological systems. This event, similar to proliferation, is tightly regulated with both processes playing essential roles in the homeostasis of renewable tissues. If apoptosis is under inappropriate control, various diseases such as cancer, autoimmune diseases and neurodegenerative diseases are caused (Kerr et al, Br. J. Cancer, 16, 239–257, 1972). Apoptosis is characterized by morphological and biochemical changes, causing the damage of cell junctions in cells, nuclear membrane blebbing, and cell shrinkage in the early stage, and chromatin condensation, cytoplasmic and nuclear condensation, mitochondrial membrane potential loss, the change of cytoplasmic membrane compositions, and the formation of apoptotic bodies in the late stage. As a final event of the cells undergoing apoptosis, internucleosomal DNA degradation into nucleosomal fragments is the most dramatic and fatal biochemical feature (Green D. R., and Reed, J. C., Science, 281, 1309–1312, 1998).
Extensive research has revealed key players and mechanisms involved in apoptosis in various organisms. For example, nematodes such as Canorhabdtis elegans are reported to have three molecules playing important roles in controlling apoptosis: CED-3, CED-4 and CED-9. Both of CED-3 and CED-4 are indispensable for the induction of apoptosis, while CED-9, which shares amino acid sequence homology with mammalian Bcl2 family proteins, acts as an inhibitor of CED-4, which is the nematode homologue of the mammalian Apaf-1. Particularly, CED-3 is known to have the same function as that of caspase family proteins of cystein, aspartate specific proteases.
Execution of apoptosis is mediated by members of the caspase family. After an apoptotic stimulus is received, the zymogen form of the caspase is rapidly activated by proteolytic processing at highly related aspartic acid cleavage sites to generate the heteromeric subunits of the active protease. Once activated by various apoptotic stimuli, the caspase family of proteins decomposes cellular proteins such as RAPD (poly(ADP-ribose) polymerase), lamine, cytokeratins, and inhibitor of caspase-activated deoxyribonuclease (ICAD) to mediate apoptosis (Cryns, V., and Yuan, J., Genes Dev., 12, 1551–1570, 1998). Conditions associated with the apoptosis mediation of caspases include receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage, and disruption of cell—cell adhesion or cell-matrix interaction. As a result of the active research directed to the caspase family, at least 14 different caspases have been found in mammalian cells. Basically, caspases are cysteine proteases that cleave after aspartic acid residues, which are required for the initiation and execution of apoptosis. That is, caspases have an absolute requirement in the target substrate sequence with cleavage occurring at the carbonyl end of the aspartic acid residue (Nunez et al., Oncogene, 17, 3272–3245, 1998).
DNA fragmentation, a hallmark of apoptosis, is an irreversible event closely associated with caspase activation. In the cells undergoing apoptosis, nuclear DNA is degraded into DNA fragments of 180–200 bp in size by endogenous endonucleases, which show a ladder-like pattern when run on an agarose gel. Functioning as the endogenous endonuclease, CAD (caspase-activated DNase), also called CPAN (caspase activated DNase) and DFF (DNA fragmentation factor), has been recently isolated from human and murine cells (Liu et al., Proc. Natl. Acad. Sci. U.S.A., 95, 1841–1846, 1998; Liu et al., Cell, 89, 175–184, 1997). DFF is a heterodimeric protein complex consisting of DFF45 (molecular weight 45 kDa) and DFF40 (molecular weight 40 kDa). Purified DFF40 alone possesses intrinsic nuclease activity, which is inhibited by its association with DFF45. The proteolytic cleavage of DFF45 by caspase-3 frees DFF40 to function as a nuclease. Mouse homologue of human DFF was identified as a DNase inhibitor designated ICAD, for inhibitor of caspase-activated DNase. As in DFF, CAD remains inactive while being associated with ICAD. Upon cleavage of ICAD, a caspase activated deoxyribonuclease (CAD) is released, activated and eventually causes the degradation of DNA in the nuclei. Therefore, ICAD, which consists of 331 amino acids, is the mouse counterpart of the human DFF45. ICAD has been recently found to exist in a long form (ICAD-L) and a short form (ICAD-S). Two different ICAD cDNAs (ICAD-L and ICAD-S) were isolated from a mouse T-cell lymphoma cDNA library. These cDNAs contained open reading frames of 331 and 265 amino acids, respectively. The two cDNAs were identical up to amino-acid position 261, after which the sequences differ (ICAD-S has four characteristic C-terminal amino acid residues), suggesting that the ICAD-L and ICAD-S messenger RNAs are generated through alternative splicing (Sakahira et al., J. Biol. Chem., 274, 15740–15744, 1998).
CAD/DFF40 is produced as a complex with ICAD/DFF45: treatment with caspase-3 releases the DNase activity that causes DNA fragmentation in nuclei. ICAD/DFF45 therefore seems to serve as a molecular chaperone for inducing an appropriate folding of CAD/DFF40 during its synthesis, as well as functioning as a selective inhibitor of CAD/DFF40. CAD/DFF40 is inactive while being complexed with ICAD/DFF45. The inhibition of the nuclease activity of CAD/DFF40 by ICAD/DFF45 can be broken by caspase-3: cleavage of ICAD/DFF45 at both Asp positions 117 and 224 by caspase-3 inactivate ICAD/DFF45, allowing CAD/DFF40 to be active as an endonuclease (Mcllroy et al., Oncogene, 18, 4401–4408, 1999). Of the caspases identified so far, caspase-3 and caspase-7 are known to inactivate ICAD/DFF45 by cleavage in vitro. However, caspase-7 is reported to be unable to produce the active form of CAD/DFF40: caspase-3 is regarded as an important mediator for CAD-dependent DNA fragmentation (Wolf et al., J. Biol. Chem., 274, 30651–30656, 1999). Another group involved in the activation of CAD/DFF40 is exemplified by CIDEs whose N-terminal region CIDE-N is found to be conserved in ICAD/DFF45 (Inhohara et al., EMBO. J., 17, 2526–2533, 1998).
There exist MAPK (mitogen-activated protein kinase) signaling pathways that are closely related to apoptotic cell death (Cobb, M. H., and Go.dsmith, E. J., J. Biol. Chem., 270, 14843–14846, 1995; Dickens et al., Science, 277, 693–696, 1997). Triggered by extracellular sitimuli, MAPK signaling pathways are known to control a number of genes taking part in cell cycle control. Typically, a well-defined three-kinase module composed of a MAPK, a MKPK kinase (MAPKK) and a MAPKK kinase (MAPKKK), which act in a cascade pattern, is involved in each MAPK signaling pathway. In the MAPK cascade, MAPKKK is activated by an upstream kinase or an adaptor protein and then phosphorylates MAPKK which then becomes its active form, leading to MAPK activation. Activated MAPK moves into the nucleus and phosphorylates downstream kinases, thereby it regulates gene expression or other cellular events.
At least three MAPK cascades have been identified in mammals, each consisting of the three-kinase module. These cascades play key roles in relaying various physiological, environmental or pathological signals from the extracellular environment to the transcriptional machinery in the nucleus, for which extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase/stress-activated protein kinase, and p38 MAPK are responsible, respectively (Boulton et al., Cell, 65, 663–675, 1991; Cobb, M. H., and Goldsmith, E. J., J. Biol. Chem., 270, 14843–14846, 1995; Fringer et al., Curr. Opin. Genet. Dev., 7, 67–74, 1997; Han et al., Science, 265, 808–811, 1994).
Before playing a role in growth factor-mediated activation and differentiation, ERK is activated in cells upon stimulation with mitogenic factors, such as insulin, epidermal growth factor (EGF), and phorbol ester. One of the MAPKs, JNK stimulates the transcriptional activity of c-Jun preferentially in response to proinflammatory cytokines, and certain environmental stresses, such as UV light, heat shock, or osmotic stress (Ahmed, A., and Salahuddin, A., Indian. J. Biochem. Biophys., 31, 156–159, 1994; Derijard et al., Science, 267, 682–685, 1994; Galcheva-Gargova et al., Science, 265, 806–808, 1994). Activation of p38 kinase, which is highly homologous to yeast HOG1 kinase, is induced by high osmotic pressures, lipopolysaccharide, TNF-α, and interleukin-1 (Han et al., Science, 265, 808–811, 1994; Rouse et al., Cell, 78, 1027–1037, 1994).
Of the signaling pathways, JNK and p38 cascades are suggested to play important roles in the apoptotic cell death caused by various stresses (Butterfield et al., J. Biol. Chem., 272, 10110–10116, 1997; Kawadaki et al., J. Biol. Chem., 272, 18518–18521, 1997). Three jnk genes, jnk1, jnk2 and jnk3, have been isolated thus far (Gupta et al., EMBO. J., 15,2760–2770, 1996; Widmann et al., Physiol. Rev. 79, 143–180, 1999) and are found to be activated by two MAPKKs, MKK4/SEK1 (Derijard et al., Cell, 76, 1025–37, 1999) and MKK7 (Holland et al., J. Biol. Chem., 272, 24944–24948, 1997). As for p38 kinase, its four genes, p38a, p38b, p38c and p38d, were cloned (Gpedert et al., EMBO. J., 16, 3563–357, 1997; Han et al., Science, 265, 808–811, 1994; Jiang et al., J. Biol. Chem., 272, 30122–30128, 1997; Lee et al., Nature, 272, 23668–23674, 1994; Wang et al., J. Biol. Chem., 272, 23668–23674, 1997) and all are activated by MKK3 and MKK6. Various MAPKKKs have been identified as upstream activators in the JNK and p38 cascades. Of them, TAK1 (tumor growth factor-activated kinase 1) activates both of the JNK and p38 cascades (Gerwins et al., J. Biol. Chem., 272, 8288–8285, 1997; Takekawa et al., EMBO. J., 16, 4973–4982, 1997; Yamaguchi et al., Science, 270, 2008—2008–2011, 1995).
Also, a novel MAPKKK was recently identified and designated as ASK1 (for apoptosis signal-regulating kinase 1). ASK1 is reported to activate two different subgroups of MAPKK, SEK1/MKK4 and MKK3/MAPKK6/MKK6, which in turn activate stress-activated protein kinase (JNK) and p38 MAP kinase, respectively (Ichijo et al., Science, 275, 90–94, 1997; Yamaguchi et al., Science, 270, 2008–2011, 1996). That is, ASK1 activates the SEK1-JNK pathway and the MKK3/6-p38 pathway, selectively. In addition to the apoptosis caused by stresses such as H2O2, UV light and other DNA-damaging agents, ASK1 plays an important role in TNFα- and Fas-induced apoptosis, according to previous reports (Hsu et al., Immunity, 4, 387–396, 1996; Rothe et al., Cell, 83, 1243–1252, 1994; Shu et al., Proc. Natl. Acad. Sci. U.S.A., 93, 13973–13978, 1996). ASK1 is activated by TNFR and Fas through the interaction between the C-terminal non-catalytic region of ASK1 and the conserved C-terminal TRAF domain of the TRAF family TRAF2 (Hoeflich et al., Oncogene, 18, 5814–5820, 1999) and through the interaction with Fas-associated protein Daxx (Chang et al., Science, 281, 1860–1863, 1998). The Daxx-induced ASK1 activation stimulates both the JNK and p38 pathways, leading to apoptotic cell death (Chang et al., Science, 281, 1860–1863, 1998; Yang et al., Cell, 89, 1067–1076, 1997). ASK1, therefore, acts as a common mediator of TNFα- and Fas-induced signal cascades and study on proteins and molecular mechanisms controlling the activity of ASK1 will help understand apoptosis processes better.
Leading to the present invention, the intensive and thorough research on the ASK1-mediated apoptosis pathway, conducted by the present inventors, resulted in the finding that CAD is inhibited by a mouse protein that can interact with ASK1. The protein, named CAD inhibitor interacting with ASK1 (abbreviated to “CIA”), is found to directly interact with ASK1 to inhibit ASK1-mediated apoptosis. In addition, CIA is identified to bind to CAD to inhibit CAD-mediated DNA fragmentation. Therefore, the novel CIA gene or its protein functions as a selective inhibitor of CAD- and ASK1-mediated apoptosis. Based on its intracellular functions, the CIA gene or protein can be used as a therapeutic agent for treatment of degenerative diseases of the cranial nervous system, apoplexy, cancer, immune disorders and inflammation.